Crosslinking of biological molecules is often desired for optimum effectiveness in biomedical applications. For example, collagen, which constitutes the structural framework of biological tissue, has been extensively used for manufacturing bioprostheses and other implanted structures, such as vascular grafts, wherein it provides a good medium for cell infiltration and proliferation. However, biomaterials derived from collagenous tissue must be chemically modified and subsequently sterilized before they can be implanted in humans. The fixation, or crosslinking, of collagenous tissue increases strength and reduces antigenicity and immunogenicity.
Collagen sheets are also used as wound dressings, providing the advantages of high permeability to water vapor and rapid wound healing. Disadvantages include low tensile strength and easy degradation of collagen by collagenase. Crosslinking of collagen sheets reduces cleavage by collagenase and improves tensile strength.
Clinically, biological tissue has been used in manufacturing heart valve prostheses, small-diameter vascular grafts, and biological patches, among others. However, the biological tissue has to be fixed with a crosslinking or chemically modifying agent and subsequently sterilized before they can be implanted in humans. The fixation of biological tissue is to reduce antigenicity and immunogenicity and prevent enzymatic degradation. Various crosslinking agents have been used in fixing biological tissue. These crosslinking agents are mostly synthetic chemicals such as formaldehyde, glutaraldehyde, dialdehyde starch, glyceraldehydes, cyanamide, diimides, diisocyanates, and epoxy compound. However, these chemicals are all highly cytotoxic which may impair the biocompatibility of biological tissue. Of these, glutaraldehyde is known to have allergenic properties, causing occupational dermatitis and is cytotoxic at concentrations greater than 10–25 ppm and as low as 3 ppm in tissue culture. It is therefore desirable to provide a crosslinking agent suitable for use in biomedical applications that is within acceptable cytotoxicity and that forms stable and biocompatible crosslinked products.
To achieve this goal, a naturally occurring crosslinking agent (genipin) has been used to fix biological tissue. The co-pending application Ser. No. 09/297,808 filed Sep. 27, 2001, entitled “Chemical modification of biomedical materials with genipin” is incorporated and cited herein by reference. The cytotoxicity of genipin was previously studied in vitro using 3T3 fibroblasts, indicating that genipin is substantially less cytotoxic than glutaraldehyde (Sung H W et al., J Biomater Sci Polymer Edn 1999; 10:63–78). Additionally, the genotoxicity of genipin was tested in vitro using Chinese hamster ovary (CHO-K1) cells, suggesting that genipin does not cause clastogenic response in CHO-K1 cells (Tsai C C et al., J Biomed Mater Res 2000; 52:58–65). A biological material treated with genipin resulting in acceptable cytotoxicity is key to biomedical applications.
It is further hypothesized in the literature that acellular tissue might remove cellular antigens (Wilson G J et al., Trans Am Soc Artif Intern 1990; 36:340–343). As a means for reducing the antigenic response to xenograft material, cell extraction removes lipid membranes and membrane-associated antigens as well as soluble proteins. Courtman et al. developed a cell extraction process to render bovine pericardium free of cells and soluble proteins, leaving a framework of largely insoluble collagen and elastin (Courtman D W et al., J Biomed Mater Res 1994; 28:655–666). They hypothesized that this process may decrease the antigenic load within the material, reducing the associated degradation due to in vivo cellular attack, and possibly eliminating the need for extensive crosslinking. Additionally, acellular tissue may provide a natural microenvironment for host cell migration to accelerate tissue regeneration (Malone J M et al., J Vasc Surg 1984; 1:181–91).
Other than maintaining a natural microenvironment, the collagen matrix, including soluble collagen, after being treated with the proposed cell extraction process, the collagen matrix shall have similar properties of decreased antigenicity/immunogenicity. However, the framework of largely insoluble collagen and elastin matrix is still vulnerable to enzymatic degradation and is not suitable as an implantable bioprosthesis.
As is well known that the human knee comprises an articulation of the femur, the tibia and the patella. The femur and the tibia are maintained in a condition of stable articulation by a number of ligaments of which the principal ones are the anterior and posterior cruciate ligaments and the collateral ligaments. The rupture of the anterior cruciate ligament is relatively commonly encountered as a result of sporting injury or the like. This rupture leads to knee instability and can be a debilitating injury. Though less common, the rupture of the posterior cruciate ligament can be equally disabling.
In the past, polymer or plastic materials have been studied as ligament or tendon replacements. Prosthetic ligament replacements made of carbon fibers and Gore-Tex PTFE materials do not last a long period of time. Repeated loading of a prosthetic ligament in a young active patient leads to failure of the ligament. It has been found that it is difficult to provide a tough durable plastic material which is suitable for long-term connective tissue replacement. Plastic material could become infected and difficulties in treating such infections often lead to graft failure.
In accordance with the present invention, there is provided genipin treated tissue grafts for orthopedic and other surgical applications, such as vascular grafts and heart valve bioprostheses, which have shown to exhibit many of the desired characteristics important for optimal graft function. In particular, the tissue regeneration capability in the genipin-fixed acellular tissue may be suitable as a graft material for bone, tendon, ligament, cartilage, muscle, and cardiovascular applications.
In some aspects of the invention, it is provided a method for promoting autogenous ingrowth of a biological tissue material, comprising providing a natural tissue, removing cellular material from the natural tissue, increasing porosity of the natural tissue by at least 5%, loading an angiogenesis agent or autologous cells into the porosity, and crosslinking the natural tissue with a crosslinking agent.